Volume 26, Issue 3, Pages 507-517.e3 (January 2019) Synapse Formation Activates a Transcriptional Program for Persistent Enhancement in the Bi-directional Transport of Mitochondria  Kerriann K. Badal, Komol Akhmedov, Phillip Lamoureux, Xin-An Liu, Adrian Reich, Mohammad Fallahi-Sichani, Supriya Swarnkar, Kyle E. Miller, Sathyanarayanan V. Puthanveettil  Cell Reports  Volume 26, Issue 3, Pages 507-517.e3 (January 2019) DOI: 10.1016/j.celrep.2018.12.073 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2019 26, 507-517.e3DOI: (10.1016/j.celrep.2018.12.073) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Synapse Formation Produces Persistently Enhanced Bi-directional Mitochondrial Transport in Pre-synaptic Sensory Neurons (A) Experimental design. (B) DIC and fluorescence images are shown. The fluorescence images in boxes show examples of a region where transport was analyzed. MN, motor neuron; NOC, nocodazole; SN, sensory neuron. Scale bar, 10 μm. (C–F) Bar graphs show the flux and velocity of anterograde (Ant) and retrograde (Ret) mitochondrial transport in SN measured by kymograph analysis. Error bars are SEMs. NS, nonsignificant, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. One-way ANOVA and Tukey post hoc test. (G–I) Bar graphs show the flux and velocity of anterograde and retrograde transport in SN compared to SN co-cultured with L7MN at 6, 12, and 24 h after plating, respectively. Numbers of neurons analyzed are indicated in the bar graphs. Error bars are SEMs. NS, nonsignificant; ∗p < 0.05. Student’s unpaired t test. See also Figure S1 and Table S1A. Cell Reports 2019 26, 507-517.e3DOI: (10.1016/j.celrep.2018.12.073) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Activation of cAMP Signaling but Not IP3R Nor PKC Signaling Is Sufficient for Enhanced Mitochondrial Transport (A) Experimental design. (B–G) Bar graphs showing flux and velocity of anterograde and retrograde mitochondrial transport in SN measured in the presence of PMA, ADA, forskolin (FK), or FK+ PKAi (PKA inhibitor). (H) Kymograph analysis following exposure to different pharmacological agents. (I) Bar graph shows total mitochondrial density, which includes both stationary and transported mitochondria. The numbers of neurons analyzed are indicated in bar graphs. Error bars are SEMs. NS, nonsignificant; ∗p < 0.05; ∗∗ p < 0.05. Student’s unpaired t test. See also Figure S2 and Table S1B. Cell Reports 2019 26, 507-517.e3DOI: (10.1016/j.celrep.2018.12.073) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Role of NMDA and AMPA Receptor Signaling in Modulating Pre-synaptic Mitochondrial Transport (A) Experimental design. (B) Representative traces of 2 s recording before and after CNQX or APV. (C) Bar graphs showing the quantitation of data in (B). (D–I) Bar graphs showing the quantitation of flux and velocity of anterograde and retrograde transport at different time points: (D) 15 min CNQX; (E) 30 min APV; (F) 1 h CNQX; (G) 1 h APV; (H) 24 h CNQX and APV flux; (I) 24 h CNQX and APV velocity. The numbers of neurons used in the experiment are indicated in the bar graphs. Error bars are SEMs. NS, nonsignificant; ∗p < 0.05; ∗∗∗p < 0.001. Student’s unpaired t test. See also Table S1C. Cell Reports 2019 26, 507-517.e3DOI: (10.1016/j.celrep.2018.12.073) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Role of Post-synaptic Neurons in Modulating Pre-synaptic Mitochondrial Transport (A) Experimental design. (B–F) Schematic of SN-L7MN microdissection. CB, L7MN with intact cell body. CBR, L7MN with cell body removed. Bar graphs show the measurements of the flux and velocity of anterograde and retrograde transport. Mitochondrial imaging was done on the SN 24 h or 72 h after removal of the L7MN cell body (C and D, respectively) and following exposure to (E) actinomycin D (Act) or (F) anisomycin (Ani). Numbers of neurons used in the experiment are indicated in the bar graphs. Error bars are SEMs. NS, nonsignificant; ∗p < 0.05; ∗∗p < 0.01. Student’s unpaired t test. (G) Schematics of the experiment for microarray analysis. SNCBs were isolated from SN grown alone or co-cultured with L7MN (DIV 4–6). SN CB, sensory neuron cell body. (H) Heatmap showing differential gene expression (red, upregulated; green, downregulated; 2-fold cutoff; FDR <0.05) in SN co-cultured with L7MN (n = 4) or SN alone (n = 6). (I) Pie chart representing microarray data showing pathways that are enriched upon synapse formation. (J) Bar graph shows fold change in transcripts that are enriched with synapse formation within the pre-synaptic SN. Fold increase in KHC (kinesin heavy chain), DLC (dynein light chain), and dynactin. qPCR validation of three genes that we identified from microarray analysis. Bar graphs show normalized fold changes in gene expression in SN-L7MN compared to SN alone. 18srRNA expression was used to normalize data. Error bars are SEMs; ∗p < 0.05; Student’s t test. (K and L) Summary of findings. Persistently enhanced mitochondrial transport through changes in the pre-synaptic transcriptional program. Positive, negative, and neutral changes in mitochondrial transport identified from our studies are shown using emojis. Arrows, bi-directional transport; black squiggly lines, differentially expressed transcripts; red squiggly lines with beads, polyribosomes. SN, pre-synaptic sensory neuron; MN, post-synaptic motor neuron. See also Figures S3 and S4 and Tables S1D and S1H–S1K. Cell Reports 2019 26, 507-517.e3DOI: (10.1016/j.celrep.2018.12.073) Copyright © 2018 The Author(s) Terms and Conditions